Three-dimensional reconstruction and motion analysis of living, crawling cells

Cell behavior is three-dimensional (3-D), even when it takes place on a flat surface. Migrating cells form pseudopods on and off the substratum, and the cell body undergoes height changes associated with a 1 min behavior cycle. Inside the cell, the nucleus has a 3-D migratory cycle, and vesicles move up and down in the z-axis as a cell locomotes. For these reasons, the two-dimensional (2-D) analysis of cellular and subcellular behavior is, in many cases, inadequate. We have, therefore, developed 3-D motion analysis systems that reconstruct the cell surface, nucleus, pseudopods, and vesicles of living, crawling cells in 3-D at time intervals as short as 1 s, and compute more than 100 parameters of motility and dynamics morphology at 1-s intervals. We are now in the process of developing a multimode reconstruction system that will allow us to reconstruct and analyze fluorescently tagged molecular complexes within the differential interference contrast-imaged subcellular architecture of a crawling cell. These evolving technologies should find wide application for a host of biomedical problems.